Mutants of glycoside hydrolases and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates

Abstract

Method for preparing the disaccharide [α-D-Gldp(1→3)]-α-L-Rhap-YR wherein Y is selected from —O— and —S— and R is selected from the group consisting of: C 1 -C 6 alkyl, C 1 -C 6 alkenyl, aryl, allyl, —CO-alkyl (C 1 -C 6 ), —CO-alkenyl (C 1 -C 6 ), —CO-aryl comprising the step of using a mutant of a wild type glycoside hydrolase.

1 .- 14 . (canceled) 
     
     
         15 . A method for preparing the disaccharide [α-D-Glcp(1→3)]-α-L-Rhap-YR of formula (Ia), wherein Y is selected from —O— and —S— and R is selected from the group consisting of: C 1 -C 6  alkyl, C 1 -C 6  alkenyl, aryl, allyl, —CO-alkyl (C 1 -C 6 ), —CO-alkenyl (C 1 -C 6 ), —CO-aryl: 
       
         
           
           
               
               
           
         
         said method comprising the step of reacting:
 A) a mutant of a wild type glycoside hydrolase, 
 wherein said wild type glycoside hydrolase has 450 to 850 amino acids and comprises from the N- to C-terminus eleven motifs defined by the following consensus sequences: 
 (1) the amino acid sequence LGVNYLHLMPL (SEQ ID NO: 1), which is located in the β-strand 2 of said wild type glycoside hydrolase; 
 (2) the amino acid sequence DGGYAV (SEQ ID NO: 2), which is located in the loop 2 of the (β/α) 8 -barrel of said wild type glycoside hydrolase; 
 (3) the amino acid sequence DFVFNH (SEQ ID NO: 3) which is located in the β-strand 3 of said wild type glycoside hydrolase; 
 (4) the amino acid sequence LREIFPDTAPGNF (SEQ ID NO: 4), which is located in the domain B of said wild type glycoside hydrolase; 
 (5) the amino acid sequence FNSYQWDLN (SEQ ID NO: 5), which is located in the C-terminal part of the domain B of said wild type glycoside hydrolase; 
 (6) the amino acid sequence ILRLDAVAFLWK (SEQ ID NO: 6), which is located in the β-strand 4 of said wild type glycoside hydrolase; 
 (7) the amino acid sequence EAIV (SEQ ID NO: 7), which is located in the β-strand 5 of said wild type glycoside hydrolase; 
 (8) the amino acid sequence YVRCHDDI (SEQ ID NO: 8), which is located in the β-strand 7 of said wild type glycoside hydrolase; 
 (9) the amino acid sequence RISGTLASLAG (SEQ ID NO: 9), which is located in the domain B′ of said wild type glycoside hydrolase; 
 (10) the amino acid sequence GIPLIYLGDE (SEQ ID NO: 10), which is located in the β-strand 8 of said wild type glycoside hydrolase; 
 (11) the amino acid sequence RWVHRP (SEQ ID NO: 11), which is located in the loop 8 of the (β/α) 8 -barrel, 
 and the sequence formed by said eleven motifs joined end-to-end from motif (1) to motif (11) of said wild type glycoside hydrolase has at least 65% sequence identity or at least 80% sequence similarity with the amino acid sequence SEQ ID NO: 12; 
 wherein said mutant has a mutation consisting of: 
 the substitution of the amino acid residue at position 4 in said motif (4) with any amino acid selected from the group consisting of alanine (A), cysteine (C), glycine (G), histidine (H), lysine (K), asparagine (N), arginine (R), serine (S), threonine (T), tryptophan (W) and tyrosine (Y), or 
 the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of cysteine (C), glutamic acid (E), isoleucine (I) and valine (V), 
 B) with the acceptor of formula (IIa): 
 
       
       
         
           
           
               
               
           
         
         
           wherein Y and R are defined as above, and 
           C) with a donor of formula (IIIa): 
         
       
       
         
           
           
               
               
           
         
         
           wherein R 1  represents a group selected from: 
         
       
       
         
           
           
               
               
           
         
       
     
     
         16 . A method according to  claim 15 , wherein said wild type glycoside hydrolase is an amylosucrase (EC 2.4.1.4) or sucrose hydrolase (EC 3.2.1.-). 
     
     
         17 . A method according to  claim 16 , wherein said wild type glycoside hydrolase is an amylosucrase from  Neisseria polysaccharea,  and is preferably selected from the group consisting of 1G5A (SEQ ID NO: 13), 1ZS2, 1MVY, 1MW0, 1S46, 1JGI, 1MW2, 1MW3, 1MW1 and 1JG9 proteins. 
     
     
         18 . A method according to  claim 15  for preparing the disaccharide [α-D-Glcp(1→3)]-α-L-Rhap-OMe of formula (I): 
       
         
           
           
               
               
           
         
         comprising the step of reacting a mutant of a wild type glycoside hydrolase as defined in  claim 15 , with the acceptor of formula (II) 
       
       
         
           
           
               
               
           
         
         and with a donor of formula (IIIa): 
       
       
         
           
           
               
               
           
         
       
     
     
         19 . A method according to  claim 15 , wherein the acceptor of formula (IIIa) is sucrose. 
     
     
         20 . A method for the preparation of the building block corresponding to the disaccharide of formula (XX 1 ): 
       
         
           
           
               
               
           
         
         wherein said method comprises at least one step according to  claim 15 . 
       
     
     
         21 . A method for preparing the disaccharide of formula (XX 1 ), the disaccharide of formula (XX 2 ), the disaccharide of formula (XX 2B ), the disaccharide of formula (XX 3 ), the disaccharide of formula (XX 3A ), the disaccharide of formula (XX 3B ), the disaccharide of formula (XX 4 ), the disaccharide of formula (XX 5 ), the disaccharide of formula (XX 5A ), the disaccharide of formula (XX 5B ), the disaccharide of formula (XX 5C ), the disaccharide of formula (XX 5D ), or the disaccharide of formula (XX 5E ): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein said method comprises at least one step according to  claim 15 . 
       
     
     
         22 . A method for the preparation of the building block corresponding to the disaccharide of formula (XX 5 ): 
       
         
           
           
               
               
           
         
         said method comprising the steps according to scheme 1: 
       
       
         
           
           
               
               
           
         
         
           methyl glycosidation of L-Rhamnose, 
           enzymatic glucosylation by treatment of methyl α-L-rhamnopyranoside used as an acceptor with a mutant of amylosucrase 
           advantageously rough purification, preferably by flash chromatography 
           acetylation 
           advantageously purification 
           acetolysis 
           advantageously purification 
           anomeric bromination 
           cyclization into an orthoester preferably with allyl alcohol, 
           deacetylation, possibly under basic conditions 
           protection of the hydroxyl groups, preferentially as ethers 
           purification preferably by crystallisation 
           orthoester opening under acid catalysis to give a glycoside or hemiacetal T 
           selective deacetylation 
         
       
     
     
         23 . A mutant of a wild type glycoside hydrolase, wherein said wild type glycoside hydrolase has 450 to 850 amino acids and comprises from the N- to C-terminus eleven motifs defined by the following consensus sequences:
 (1) the amino acid sequence LGVNYLHLMPL (SEQ ID NO: 1), which is located in the β-strand 2 of said wild type glycoside hydrolase;   (2) the amino acid sequence DGGYAV (SEQ ID NO: 2), which is located in the loop 2 of the (β/α) 8 -barrel of said wild type glycoside hydrolase;   (3) the amino acid sequence DFVFNH (SEQ ID NO: 3) which is located in the β-strand 3 of said wild type glycoside hydrolase;   (4) the amino acid sequence LREIFPDTAPGNF (SEQ ID NO: 4), which is located in the domain B of said wild type glycoside hydrolase;   (5) the amino acid sequence FNSYQWDLN (SEQ ID NO: 5), which is located in the C-terminal part of the domain B of said wild type glycoside hydrolase;   (6) the amino acid sequence ILRLDAVAFLWK (SEQ ID NO: 6), which is located in the β-strand 4 of said wild type glycoside hydrolase;   (7) the amino acid sequence EAIV (SEQ ID NO: 7), which is located in the β-strand 5 of said wild type glycoside hydrolase;   (8) the amino acid sequence YVRCHDDI (SEQ ID NO: 8), which is located in the β-strand 7 of said wild type glycoside hydrolase;   (9) the amino acid sequence RISGTLASLAG (SEQ ID NO: 9), which is located in the domain B′ of said wild type glycoside hydrolase;   (10) the amino acid sequence GIPLIYLGDE (SEQ ID NO: 10), which is located in the β-strand 8 of said wild type glycoside hydrolase;   (11) the amino acid sequence RWVHRP (SEQ ID NO: 11), which is located in the loop 8 of the (β/α) 8 -barrel,   and the sequence formed by said eleven motifs joined end-to-end from motif (1) to motif (11) of said wild type glycoside hydrolase has at least 65% sequence identity or at least 80% sequence similarity with with the amino acid sequence SEQ ID NO: 12;   wherein said mutant has a mutation consisting of:   the substitution of the amino acid residue at position 4 in said motif (4) with any amino acid selected from the group consisting of lysine (K) and arginine (R), or   the substitution of the amino acid residue at position 9 in said motif (6) with any amino acid selected from the group consisting of cysteine (C), glutamic acid (E), isoleucine (I) and valine (V).   
     
     
         24 . A mutant according to  claim 23 , wherein said wild type glycoside hydrolase is an amylosucrase (EC 2.4.1.4) or sucrose hydrolase (EC 3.2.1.-). 
     
     
         25 . A mutant according to  claim 24 , wherein said wild type glycoside hydrolase is an amylosucrase from  Neisseria polysaccharea,  and is preferably selected from the group consisting of 1G5A, 1ZS2, 1MVY, 1MW0, 1S46, 1JGI, 1MW2, 1MW3, 1MW1 and 1JG9 proteins. 
     
     
         26 . A polynucleotide encoding a mutant of wild type glycoside hydrolase of  claim 23 . 
     
     
         27 . A recombinant vector comprising a polynucleotide of  claim 26 . 
     
     
         28 . A compound selected from the group consisting of: